DNA sequence for the unique sequence herpes simplex virus type 2-glycoprotein G protein and method of expressing said unique sequence of HSV-2gG

ABSTRACT

Disclosed are recombinant, synthetically and otherwise biologically produced novel proteins and polypeptides which are encoded by the DNA sequence for HSV-2 glycoprotein G (gG) or fragments of said gG sequence, particularly by the unique sequence for gG or portions of said unique sequence. The unique sequence proteins and polypeptides are serologically active, can be produced easily and safely at low cost, are useful as diagnostic reagents for HSV-2 and as vaccines against HSV-2. Further disclosed are serological assays based on such unique sequence gG proteins and polypeptides that diagnose the presence of herpes simplex virus type 2 (HSV-2) specific antibodies and can differentiate between HSV-2 and herpes simplex virus type 1 (HSV-1) specific antibodies. Such assays are useful to diagnose genital infections, to detect for exposure to HSV-2 and to screen pregnant women to protect newborns from neonatal HSV-2 infection. Also disclosed are antibodies to such gG proteins and polypeptides which are useful therapeutically, diagnostically and for affinity purification. Further, disclosed are purified and isolated DNA molecules which can be used as probes specific for HSV-2 DNA.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/129,021, filedSep. 29, 1993now abandoned, which in turn is a continutation of U.S.Ser. No. 07/832,982, filed Feb. 10, 1992, now abandoned, which furtheris a continuation of U.S. Ser. No. 07/351,740, filed May 12, 1989, nowabandoned. This application declares priority under 35 USC Section 120from those prior filed U.S. applications.

FIELD OF THE INVENTION

This invention is in the fields of biochemical engineering andimmunochemistry. More particularly, this invention relates torecombinant DNA molecules expressed in appropriate host organisms aswell as novel proteins and polypeptide fragments thereof which may beproduced recombinantly, synthetically or by other means, such as by, thefragmentation of biologically produced proteins and polypeptides. Therecombinant DNA molecules of this invention are characterized by the DNAwhich codes for proteins and polypeptides from the herpes simplex virustype 2 (HSV-2) glycoprotein G (gG). More specifically, said DNA is thatfrom the unique sequence of glycoprotein G (gG) of HSV-2 or portions ofsaid unique sequence which code for serologically active novel proteinsand polypeptides. Said DNA can be used as probes specific for HSV-2 DNA,and said serologically active proteins and polypeptides are useful asreagents for the immunological detection of HSV-2, enabling adiagnostician to differentiate between HSV-2 type-specific antibodiesand herpes simplex type 1 (HSV-1) type-specific antibodies. Theexpressed or synthetically or biologically produced proteins andpolypeptides of this invention are further useful for the production ofantibodies and as vaccines for HSV-2.

BACKGROUND OF THE INVENTION

The herpes viruses include the herpes simplex viruses, comprising tworelated variants designated types 1 (HSV-1) and 2 (HSV-Z). Many of thecounterpart gene products of HSV-1 and HSV-2 have similar molecularweights and common antigenic determinants. However, clinicalmanifestations of HSV-1 and HSV-2 differ significantly. Nongenitalherpes infections such as common cold sores are primarily caused byHSV-1. Genital and neonatal infections are most usually associated withHSV-2. About 90% of primary genital HSV infections and about 99% ofrecurrent genital HSV infections are caused by HSV-2. [Sullender et al.,J. Infect. Dis. 157(1):164-171 (Jan. 1988).]

As estimated by physician consultations, the incidence of symptomaticgenital herpes is steadily increasing in the United States, developingamong hundreds of thousands of Americans every year. [Sullender et al.,J. Infect. Dis. 157(1): 164-167 (Jan. 1988); Tapley et al. (eds.), TheColumbia University College of Physicians and Surgeons Complete HomeMedical Guide (1985).] The prevalence of asymptomatic HSV-2 infectionshas been difficult to determine because of the strongcross-neutralization between HSV-1 and HSV-2 and because of the highincidence of antibody to HSV-1 in the population. [Plummer, Cancer Res.,33: 1469-1476 (June 1973).] As Lee et al. [J. Clin. Microbiol., 22(4):641-644 at 643 (1985)] point out: "[A] serological assay that can detectHSV-2 antibodies would be of particular epidemiological assistance.However, because of the existence of many common antigens in HSV-1 andHSV-2, specificity of the assay has been a major problem." [Emphasisadded; citation omitted.] Specificity of such an assay is importantbecause of the implications of HSV-2 infections both at theepidemiological level, for example, the relation of genital herpes tocervical cancer, and at the individual level, for example,false-positive results can lead to great problems such as impropermedical management for pregnant women or undue psychological trauma inpatients and their consorts. [Lee et al., id. at 643.] The instantinvention provides for a specific serological assay to differentiateaccurately and definitively between HSV-1 and HSV-2 antibodies.

During the past 20 years, the incidence of neonatal HSV-2 infection hasincreased significantly, paralleling the increased incidence of genitalHSV-2 infection in pregnant females--approximately a nine-fold increasefrom 1966 to 1979. [Hampar et al., U.S. Pat. No. 4,764,459 (Aug. 16,1988).] A systemic HSV-2 infection in a newborn can cause seriousproblems, including blindness, neurological problems, mental retardationand even death. The major source of neonatal HSV-2 infection is viacontact with the infected genital tract of the mother at the time ofdelivery. [Corey et al., Ann. Int. Med., 98:958-972 (1983).]Transmission of HSV-2 from mother to infant can occur during symptomaticor asymptomatic maternal infection. [Corey et al., id.] Studies haveindicated that over 70% of infants with neonatal HSV-2 infection hadmothers who were asymptomatic at the time of delivery. [Whitley et al.,Pediatrics, 66:495-501 (1980).] The overall risk of neonatal infectionhas been estimated to be about 10% in women with primary or recurrentHSV-2 infection after 8 months' gestation and 40% if the HSV-2 virus ispresent at the time of delivery. [Douglas, "DNA Viruses: Herpetoviridae"in Principles and Practice of Infectious Diseases (2d Ed.) (Mandrell etal., eds. (1985).]

The recommended procedure for a pregnant woman suspected of harboring aHSV-2 infection is to perform weekly tissue culture confirmation teststo determine whether HSV-2 is being released into the birth canal. Suchprocedures are costly and, further, the tissue culture tests are limitedby the timing of taking the samples, that is, only at certain pointsduring the infectious cycle are samples containing live virusobtainable; for example, live virus is obtainable during the activelyshedding stage in the cycle but not later when the viral vesicles aredrying. [See Spruance et al. Infect. Immun. 36(3):907-910 (June 1982);and Spruance et al., N. Engl. J. Med. 297(2): 69-75 (Jul. 14, 1977).]Therefore, the use of tissue culture tests is limited to the high riskcategory of pregnant women who show either a) a history of recurrentgenital HSV-2 infection, b) active disease during pregnancy, or c)sexual partners with proven HSV-2 infection. If an active infection isapparent, a cesarean delivery, with its associated risks, is performedas a protective measure for the baby. [Hampar et al., supra]. However,as noted above, most infants with neonatal infection due to HSV-2 areborn to mothers with no history of genital herpes. Because asymptomaticintrapartum shedding of HSV-2 from the mother's cervical or vulval areasappear to be an important source of neonatal infection, a rapid,reliable and inexpensive serological screening test to identify pregnantwomen potentially harboring HSV-2 is needed. [Corey et al., supra]. Thisinvention provides for such a screening test.

The serological assays of this invention are based upon recombinantly,synthetically or biologically produced proteins and polypeptidesspecific for HSV-2 antibodies. Such proteins and polypeptides areencoded by a unique DNA sequence, or fragments thereof, of the envelopeprotein, glycoprotein G (gG), of HSV-2, which sequence is not found inHSV-1. McGeoch et al. [J. Gen. Virol, 68: 19-38 (1987)] identified thegene coding for gG in HSV-2, delineated its nucleotide and amino acidsequences, and pointed out (at p. 19) that the HSV-2 DNA contains "anextra sequence of about 1460 base pairs" which the HSV-1 gG gene doesnot have.

Both HSV-2 gG and HSV-1 gG have segments of 153 identical amino acids attheir carboxyl-terminal end which contain their putative transmembraneanchor domain (McGeoch et al., id.). However, HSV-2 gG contains anadditional segment of 487 unique amino acids which contain the putativetype-2 specific epitopes observed with gG, and which are coded for bythe extra "about 1460 base pairs" identified by McGeoch et al. Roizmanet al., Virology, 133: 242-247 (1984) and Marsden et al., J., Virol.,50(2): 547-554 (May 1984) independently discovered HSV-2 gG anddeveloped monoclonal antibodies to it. Roizman et al. described twomurine monoclonal antibodies that react with HSV-2 type-specificepitopes of HSV-2 gG and proved that gG was distinct from other HSV-2envelope glycoproteins, namely, gB, gC and gD.

Use of the HSV-2 gG to detect HSV-2 type-specific antibodies has beenreported by Lee et al. [J. Clin. Microbiol., 22(4): 641-644 (Oct.1985)], Sullender et al. [J. Inf. Dis., 157(1): 164-171 (Jan. 1988)],and Ashley et al. [J. Clin. Microbiol., 26(4): 662-667 (April 1988)]. Ineach of these studies, immunoaffinity purified, native, full-length,glycosylated gG was employed. Since full-length gG was used, the assayswere subject to cross-reactivity with HSV-1 antibodies in the test serabecause of the commonality of certain domains in both HSV-1 and HSV-2gG. Sullender et al. and Ashley et al. suggest the possible clinical useof the HSV-2 gG antibody assay in the diagnosis of genital infectionsand also in screening pregnant women. However, their assays, requiringthe culturing of HSV-2, isolation of the virus and affinity purifyingHSV-2 gG from viral lysate antigen preparations with monoclonalantibodies to HSV-2, are expensive to prepare and basically researchtools at this time.

Hampar et al. [U.S. Pat. No. 4,764,459 (Aug. 16, 1988)] claimsimmunoassay methods for detecting antibodies to either HSV-1 or HSV-2wherein the patients' sera are absorbed with heterologous virus-infectedcell extracts to remove intertypic cross-reacting antibodies and thenapplied to microtiter plates containing the target antigens, eitherimmunoaffinity purified HSV-1 glycoproteins (gC and/or gD) or HSV-2glycoproteins (gD and/or gF).

Markoulatos et al. [European Patent App. Pub. No. 263,025 (pub. Apr. 6,1988)] discloses antigenic glycoprotein fractions of HSV-1 and HSV-2(gC) and HSV-1 and HSV-2 (gD), purified from respectively infectedcells, and claims their use to differentiate between HSV-1 and HSV-2infections.

Su et al. [J. Virol., 62(10): 3668-3674 (Oct. 1988) report expressingHSV-2 gG in a mammalian cell line. The gG expressed was full length andglycosylated.

Burke et al. [U.S. Pat. No. 4,618,578 (Oct. 21, 1986)] claims methodsand compositions for recombinantly producing in yeast polypeptides whichare immunologically cross-reactive with glycoprotein D (gD) of HSV-1 andHSV-2. Burke et al. state (at col. 2 lines 6-9) that the "[p]roductionof gD in a yeast host provides the advantages of high levels ofexpression and modification of the polypeptides not available withprokaryotic hosts . . . "

Watson et al., [Science, 218: 381-384 (Oct. 22, 1982)], report theexpression of a HSV-1 gycoprotein D (gD) gene in Escherichia coli (E.coli). Watson et al. state that the fusion of the gD coding region withthe E. coli lac promoter enabled them to synthesize a gD-relatedpolypeptide, which when injected into rabbits elicited neutralizingantibody to both HSV-1 and HSV-2. Weis et al. [Nature, 302: 72-74 (March1983) report higher level of expression of gD in E. coli, wherein ahybrid gene encoding a chimaeric protein containing HSV-1 gD,bacteriophage lambda Cro and E. coli beta-galactosidase protein wasconstructed.

Berman et al., EP 139417 [European Pat. App. Pub. No. 139,417 (pub. Feb.2 1989)] discloses the expression of HSV-1 glycoprotein D (gD) inChinese hamster ovary cells (CHO). Claimed therein are vaccines againstHSV-1 and HSV-2 comprising at least one glycoprotein of HSV-1 or HSV-2,preferably gD or gC.

Kino et al. [U.S. Pat. No. 4,661,349 (Apr. 28, 1987)] claims a HSVsubunit vaccine effective against both HSV-1 and HSV-2 which comprises ahighly purified native glycoprotein B (gB) common to both serotypes.Cohen et al. [U.S. Pat. No. 4,762,708 (Aug. 9, 1988)] disclosesimmunologically active preparations of purified, native HSV envelopeglycoproteins, gD-1 and gD-2, useful in vaccines against HSV-1 andHSV-2.

At this time, the only commercially available means of differentiallydiagnosing a HSV-2 infection from a HSV-1 infection is by a monoclonalantibody-based tissue culture confirmation test which is relativelyexpensive compared to a blood test and time consuming, taking from atleast 24 to 72 hours. Further, such tissue culture confirmation testsare limited because of the above-noted problems associated withobtaining tissue specimens with viable virus. Further, the tissueculture confirmation tests are prohibitively expensive for use inscreening asymptomatic carriers of HSV-2. The instant invention providesa substantially cheaper, much quicker and non-time dependent method ofserologically identifying HSV-2 type-specific antibodies.

Conventional wisdom in the immunochemistry art appears to considernative glycosylation patterns of antigens important to theconformational aspects of epitopes and necessary for serotypespecificity. [See: Berman et al., Science, 222: 524-527 at 525 (Nov. 4,1983); Wilcox et al., J. Virol., 62(6): 1941-1947 (June 1988); Sugawaraet al., J. Gen. Virol., 69 (pt. 3): 537-547 (March 1988); Caust et al.,Arch. Virol., 96(3-4): 123-124 (1987); Hongo et al., Vaccine, 3(3suppl.):223-226 (Sept. 1985); Alexander et al., Science, 226 (4680):1328-1330 (Dec. 14, 1984); Wayne et al., supra at pp. 1-2; but see:Glorioso et al., Virol., 126(1): 1-18 (Apr. 15, 1983) (wherein it isstated at p. 16: "Although carbohydrate does not appear to be essentialfor maintenance of antigenicity, it cannot be ruled out that thecarbohydrate moieties may play an important role in protein conformationand that some antigenic determinant sites are formed as a consequence ofprotein secondary structure".] The instant invention controverts suchconventional wisdom in that the recombinantly produced proteins andpolypeptides of this invention which are type-specific for HSV-2antibodies can be nonglycosylated, having been expressed in aprokaryotic host.

SUMMARY OF THE INVENTION

This invention is directed to novel proteins and polypeptides encoded bythe HSV-2 gG gene or fragments thereof and to the biochemicalengineering of the HSV-2 gG gene or fragments thereof into suitableexpression vectors; transformation of host organisms with suchexpression vectors; and production of HSV-2 gG proteins and polypeptidesby recombinant, synthetic or other biological means. Such recombinant gGproteins and polypeptides can be glycosylated or nonglycosylated and canbe purified to substantial purity according to methods described herein.The invention further concerns such gG polypeptides and proteins whichare synthetically or biologically prepared. One use of such gG proteinsand polypeptides is as vaccines.

Further this invention concerns recombinant DNA molecules comprising aDNA sequence that encodes not only a HSV-2 gG protein or polypeptide butalso an amino acid sequence of a protein/polypeptide which is notimmunogenic to humans and which is not typically reactive to antibodiesin human bodily fluids. An example of such a DNA sequence is thealpha-peptide coding region of beta-galactosidase. Further, claimedherein are such recombinant fused protein/polypeptides which aresubstantially pure and non-naturally occurring.

Further, this invention concerns purified and isolated DNA moleculescomprising the unique sequence of HSV-2 gG or fragments thereof,including the nucleotide sequence from the unique sequence shown inFIGS. 1A-1B from nucleotide 45 to nucleotide 1386. Said DNA moleculescan be used as probes specific for HSV-2 DNA.

More particularly, the invention is directed to biochemical engineeringwherein the fragment of the gG gene is from the unique sequence orfragments of the unique sequence and wherein the gG proteins andpolypeptides produced are unique sequence gG proteins and polypeptideswhich similarly can be synthetically or naturally prepared.

A further aspect of this invention relates to the therapeutic anddiagnostic use of antibodies to such gG and unique sequence gG proteinsand polypeptides, as well as the use of such antibodies for affinitypurifying HSV-2 gG proteins and polypeptides.

A still further aspect of this invention relates to serological assaysfor HSV-2 type-specific antibodies employing the recombinantly,synthetically or otherwise biologically produced unique sequence gGproteins and polypeptides of this invention. This aspect of theinvention overcomes the problems of nonspecificity of previouslyreported serological assays to diagnose HSV-2 infections. Thus thepresent invention fills the needs referred to above for specificserological assays to differentiate between HSV-1 and HSV-2type-specific antibodies and for screening tests to detect from thepopulation of asymptomatic individuals, the carriers of HSV-2type-specific antibodies. Such a screening test is especially importantin relation to the population of pregnant women to avert neonatalinfections.

Such serological assays can be embodied in test kits comprising a solidphase coated with unique sequence gG proteins and polypeptides. Theinvention also provides for test kits further comprising antibodies tosuch unique sequence gG proteins and polypeptides to identify thepresence of HSV-2 in human bodily fluids, preferably vital vesiclefluid.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the nucleotide sequence for the unique sequence ofHSV-2 gG. [SEQ. ID. NO.: 1]

FIG. 2 shows the amino acid sequence which is encoded by the HSV-2 gGunique sequence. [SEQ. ID. NO.: 2]

FIG. 3 schematically outlines the construction of plasmid 19gGSE.

FIG. 4 is a map of plasmid 19gGSE.

FIG. 5 is a map of expression vector pPL-lambda [obtained from Pharmacia(Piscataway, N.J.)] useful as a source material for the preparation ofplasmid trpE/gG.

FIGS. 6A-6B schematically outline the construction of plasmid trpE/gG.

DETAILED DESCRIPTION

Definitions

The term "HSV-2 gG" refers herein to the envelope protein of HSV-2,glycoprotein G, which is encoded by a 2097 base pair gene as outlined inMcGeoch et al., supra.

The phrase "serologically active" is herein defined to mean that theprotein or polyeptide modified by that phrase are capable of detectingHSV-2 type-specific antibodies in patient samples, that is, in humanbodily fluids, including but not limited to, blood, lymph, mucous,tears, urine, spinal fluid, saliva, but most usually sera. Functionally,serological activity can be established by the immunoblot proceduresdescribed herein which are confirmed by tissue culture tests.

The phrase "unique sequence of HSV-2 gG" is herein defined to mean anucleic acid sequence coding for a portion of the envelope proteinglycoprotein G (gG) of HSV-2 which is specific for HSV-2. Said nucleicacid sequence of gG is about 1461 base pairs, coding for a sequence ofabout 486 amino acids. The nucleotide sense sequence for the uniquesequence of HSV-2 gG is shown in FIG. 1, and the amino acid sequenceencoded thereby is shown in FIG. 2. McGeoch et al. [supra at p. 19]refers to said unique sequence as "a sequence of about 1460 base pairsin the coding region of gene US4" of HSV-2 which was not found in theHSV-1 US4 gene. The phrase "unique sequence of HSV-2 gG" is hereininterpreted to include nucleotide sequences which are substantially thesame and have substantially the same biological activity as said uniquesequence of HSV-2 gG.

The phrase "gG proteins and polypeptides" is herein defined to meanproteins and polypeptides which are encoded by the HSV-2 glycoprotein GDNA sequence as outlined in McGeoch et al., supra, which article isherein incorporated by reference, or by fragments of said gG DNAsequence. The phrase "gG proteins and polypeptides" is hereininterpreted to include proteins and polypeptides which havesubstantially the same amino acid sequences and which have substantiallythe same biological activity as the "gG proteins and polypeptides".

The phrase "unique sequence gG proteins and polypeptides" is hereindefined to mean proteins and polypeptides which are encoded by theunique sequence of HSV-2 gG or fragments thereof. The phrase "uniquesequence gG proteins and polypeptides" is herein interpreted to includeproteins and polypeptides which have substantially the same amino acidsequences and which have substantially the same biological activity asthe "unique sequence gG proteins and polypeptides".

The phrase "recombinant DNA molecule" is herein defined to mean a hybridDNA sequence comprising at least two nucleotide sequences, the firstsequence not normally being found together in nature with the second.

The phrase "expression control sequence" is herein defined to mean a DNAsequence of nucleotides that controls and regulates expression ofstructural genes when operatively linked to those genes.

There are twenty main "amino acids", each of which is specified by adifferent arrangement of three adjacent DNA nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A one-letter convention is used herein toidentify said amino acids, as, for example, in FIG. 2, which conventionis outlined in Table 1.

                  TABLE 1                                                         ______________________________________                                        Amino Acid    One-Letter Symbol                                               ______________________________________                                        Alanine       A                                                               Arginine      R                                                               Asparagine    N                                                               Aspartic acid D                                                               Cysteine      C                                                               Glutamine     Q                                                               Glutamic acid E                                                               Glycine       G                                                               Histidine     H                                                               Isoleucine    I                                                               Leucine       L                                                               Lysine        K                                                               Methionine    M                                                               Phenylalanine F                                                               Proline       P                                                               Serine        S                                                               Threonine     T                                                               Tryptophan    W                                                               Tyrosine      Y                                                               Valine        V                                                               ______________________________________                                    

A "polypeptide" is a chain of amino acids covalently bound by peptidelinkages and is herein considered to be composed of 50 or less aminoacids.

A "protein" is defined herein to be a polypeptide composed of more than50 amino acids.

A "cloning vehicle" is herein defined to mean a plasmid, phage DNA orother DNA sequences which are able to replicate in a host cell,characterized by one or more endonuclease recognition sites at whichsuch DNA sequences may be cut in a determinable fashion withoutattendant loss of an essential biological function of the DNA, forexample, replication, production of coat proteins or loss of promoter orbinding sites, and which preferably contains a marker suitable for usein the identification of transformed cells, for example, tetracyclineresistance or an enzyme that effects a color change upon addition of anappropriate substrate. A cloning vehicle is alternately termed a vector.

It is understood that because of the degeneracy of the genetic code,that is, that more than one codon will code for one amino acid [forexample the codons TTA, TTG, CTT, CTC, CTA and CTG each code for theamino acid leucine (L)], that variations of the nucleotide sequence ofFIG. 1, wherein one codon is substituted for another, would produce asubstantially equivalent protein or polypeptide according to thisinvention. All such variations in the nucleotide sequence of HSV-2 gGare included within the scope of this invention.

It is further understood that the HSV-2 gG DNA unique sequence hereindescribed and shown in FIG. 1 represents only the precise structure ofthe naturally occurring nucleotide sequence. It is expected thatslightly modified nucleotide sequences will be found or can be modifiedby techniques known in the art to code for similarly serologicallyactive proteins and polypeptides, and such nucleotide sequences andproteins/polypeptides are considered to be equivalents for the purposeof this invention. DNA having equivalent codons is considered within thescope of the invention, as are synthetic DNA sequences that encodeproteins/polypeptides homologous or substantially homologous to HSV-2 gGand HSV-2 unique sequence gG proteins/polypeptides and as are DNAsequences that hybridize to the sequences coding for HSV-2 gG and uniquesequence gG proteins/polypeptides, as well as those sequences but forthe degeneracy of the genetic code would hybridize to said HSV-2 gG andunique gG sequences. Further, DNA sequences which are complementary tothe gG sequences referred to herein are within the scope of thisinvention. Such modifications and variations of DNA sequences asindicated herein are considered to result in sequences that aresubstantially the same as the HSV-2 gG and unique sequence gG sequencesoutlined herein. Typically, such related nucleotide sequences aresubstantially the same which fall into the definition of substantiallyhomologous.

Further, it will be appreciated that the amino acid sequence of HSV-2 gGcan be modified by genetic techniques. One or more amino acids can bedeleted or substituted. Such amino acid changes, especially if in aregion which is not within an epitope of the polypeptide, may not causeany measurable change in the serological activity of the protein orpolypeptide. The resulting protein or polypeptide will havesubstantially the same amino acid sequence and substantially the sameserological activity and is within the scope of the invention.

Preparation of HSV-2 gG Proteins and Polypeptides

The HSV-2 gG proteins and polypeptides of this invention, preferably theHSV-2 unique sequence gG proteins and polypeptides, can be prepared in avariety of ways according to this invention. A preferred method toprepare HSV-2 gG proteins is by recombinant means. Representativerecombinant methods of this invention are described infra under Cloningof HSV-2 gG or Fragments Thereof, Expression of HSV-2 gGProtein/Polypeptides, and Examples 1-4.

The HSV-2 gG proteins and polypeptides of this invention can further beprepared synthetically or biologically, that is, by cleaving longerproteins and polypeptides enzymatically and/or chemically. Saidsynthetic and biologic methods are described in detail infra under theheading Synthetic and Biologic Production of HSV-2 gG Protein andPolypeptide Fragments Thereof. Such methods are preferred for preparingHSV-2 gG polypeptides, as those defined under the heading Epitopes infrawhich delineate preferred serologically active regions of HSV-2 gG.

Cloning of HSV-2 gG or Fragments Thereof

The glycoprotein G of HSV-2 is encoded by a 2097 base pair gene. McGeochet al. provides the nucleotide sequence for the entire gG gene; McGeochet al. is herein incorporated by reference. A segment of said gG gene,base pairs 99-1559, is unique to HSV-2 gG, that is, said segment is notfound in the HSV-1 gG gene. A portion of the unique sequence of HSV-2gG, base pairs 143-1484, representing 92% of the unique sequence, wascloned and expressed according to this invention as shown in Example 1,below. The relationship of the aforementioned three nucleotide sequencescan be diagrammatically illustrated as follows: ##STR1##

The unique sequence of gG illustrated as base pairs 99 to 1559 above isshown herein as base pairs 1 to 1461 in FIG. 1 [SEQ. ID. NO.: 1]. Thefragment of the unique sequence cloned in Example 1 represents basepairs 45 to 1386 of FIG. 1 [SEQ. ID. NO.: 3]; said cloned sequence codesfor the amino acid sequence from amino acid 16 to amino acid 462 [SEQ.ID. NO.: 4] as shown in FIG. 2.

The plasmid 19gGSE, constructed in accordance with Example 1, is onlyrepresentational of the many possible DNA recombinant molecules that canbe prepared in accordance with this invention. Depending on therestriction endonucleases employed, all or part of the gG 2097 base pairsequence or all or part of the 1461 base pair unique sequence may becloned, expressed and used in accordance with this invention.

An appropriate starting material for isolating the HSV-2 gG gene orportions thereof is the Hind IIIl region of HSV-2 strain HG52. McGeochet al. describe at page 20 inserting said Hind IIIl fragment into theHind III site of pAT153 [commercially available from Amersham; seeBolivar et al., Gene, 2:95 (1977) and Twigg and Sherratt Nature, 283:216 (1980)] to construct a plasmid referred to in the Examples below. Analternative source for the HSV-2 gG DNA would be to digest the wholeHSV-2 DNA (isolated according to methods well known in the art) withHind III, and ligate the digested fragments into the Hind III site ofthe conmnercially available pAT53. Then an oligonucleotide of about 50base pairs may be synthesized according to methods well known in the art[for example, with an Applied Biosystems (Foster City, Calif.) DNAsynthesizer] that is complementary to an appropriate length of the HindIII fragment of interest, thereby screening the pAT153 clones for acorrect clone containing the Hind III fragment.

Useful restriction enzymes according to this invention may includeenzymes that cleave DNA in such a way that the DNA fragment generatedcontains portions of the gG unique sequence. Restriction enzymesemployed in the Examples herein include SspI, EagI, EcoRI and SmaI.Other restriction endonucleases may be similarly useful in accordancewith this invention. Their selection may be made by those of skill inthe art on due consideration of the factors set out herein withoutdeparting from the scope of the invention.

A representative cloning vehicle used in Examples 1 and 2, below, ispUC19. Said plasmid is described in Yanisch-Perron et al., Gene, 33:103(1985) and is constructed from source materials available from BethesdaResearch Laboratories. However, a wide variety of host-cloning vehiclecombinations may be usefully employed in cloning the double-strandedHSV-2 gG DNA isolated as described herein. For example, useful cloningvehicles may include chromosomal, nonchromosomal and synthetic DNAsequences such as various known bacterial plasmids such as pBR322, otherE. coli plasmids and their derivatives and wider host range plasmidssuch as RP4, phage DNA such as the numerous derivatives of phage lambda,e.g., NB989 and vectors derived from combinations of plasmids and phageDNAs such as plasmids which have been modified to employ phage DNAexpression control sequences.

Useful hosts may be eukaryotic or prokaryotic and include bacterialhosts such as E. coli strains CAG456, JM103, N4830, X1776, X2282, HB101and MRC1 and strains of Pseudomonas, Bacillus subtilis and otherbacilli, yeasts and other fungi, animal or plant hosts such as animal orplant cells in culture, insect cells and other hosts. Preferred hosts inaccordance with this invention are E. coli strains, more preferably E.coli strains CAG456, JM103 and N4830.

Of course, not all hosts may be equally efficient. The particularselection of host-cloning vehicle combination may be made by those ofskill in the art after due consideration of the principles set forthherein without departing from the scope of this invention.

Furthermore, within each specific vector, various sites may be selectedfor insertion of the isolated double-stranded DNA. These sites areusually designated by the restriction enzyme or endonuclease that cutsthem. For example, in pBR322 the PstI site is located in the gene forpenicillinase between the nucleotide triplets that code for amino acids181 and 182 of the penicillinase protein. FIG. 3 displays forillustrative purposes, some of the restriction sites in the McGeoch etal. plasmid and in pUC19.

The particular site chosen for insertion of the selected DNA fragmentinto the cloning vehicle to form a recombinant DNA molecule isdetermined by a variety of factors. These include size and structure ofthe protein or polypeptide to be expressed, susceptibility of thedesired protein or polypeptide to endoenzymatic degradation by the hostcell components and contamination by its proteins, expressioncharacteristics such as the location of start and stop codons, and otherfactors recognized by those of skill in the art. Since the expressionprocess is not fully understood, none of these factors alone absolutelycontrols the choice of insertion site for a particular protein orpolypeptide. Rather the site chosen effects a balance of these factorsand not all sites may be equally effective for a given protein.

Although several methods are known in the art for inserting foreign DNAinto a cloning vehicle to form a recombinant DNA molecule, methodspreferred in accordance with this invention are displayed in FIGS. 3 and6A-B.

Of course, other known methods of inserting DNA sequences into cloningvehicles to form recombinant DNA molecules are equally useful in thisinvention. These include, for example, direct ligation wherein the samerestriction endonuclease is employed to cleave the HSV-2 gG DNA and thecloning vehicle.

It should, of course, be understood that the nucleotide sequence or genefragment inserted at the selected restriction site of the cloningvehicle may include nucleotides which are not part of the actualstructural gene for the desired protein or may include only a fragmentof that structural gene. It is only required that whatever DNA sequenceis inserted, the transformed host will produce a protein or polypeptidedisplaying epitopes of HSV-2 gG, more preferably epitopes from theunique sequence gG which recognize type-specific HSV-2 antibodies.

The recombinant DNA molecule containing the hybrid gene may be employedto transform a host so as to permit that host (transformant) to expressthe structural gene or fragment thereof and to produce the protein orpolypeptide for which the hybrid DNA codes. The recombinant DNA moleculemay also be employed to transform a host so as to permit that host onreplication to produce additional recombinant DNA molecules as a sourceof HSV-2 gG DNA and fragments thereof. The selection of an appropriatehost for either of these uses is controlled by a number of factorsrecognized by the art. These include, for example, compatibility withthe chosen vector, toxicity of the co-products, ease of recovery of thedesired protein or polypeptide, expression characteristics, biosafetyand costs. Again, since the mechanisms of expression are not fullyunderstood, no absolute choice of host may be made for a particularrecombinant DNA molecule or protein or polypeptide from any of thesefactors alone. Instead, a balance of these factors may be struck withthe realization that not all hosts may be equally effective forexpression of a particular recombinant DNA molecule.

As indicated above, the recombinant DNA molecule may be used totransform a host so as to permit the host upon replication to produceadditional recombinant DNA molecules as a source of HSV-2 gG DNA andfragments thereof. Said DNA molecules can be purified and isolated bymethods well known in the art to create DNA probes.

When the DNA sequence for the probe is from the HSV-2 unique sequence orfragments thereof, said probes are specific for HSV-2 DNA.

Expression of HSV-2 gG Proteins/Polypeptides

Where the host cell is a procaryote such as E. coli, competent cellswhich are capable of DNA uptake are prepared from cells harvested afterexponential. growth phase and subsequently treated by the CaCl₂ methodby well known procedures. Transformation can also be performed afterforming a protoplast of the host cell.

Where the host used is an eucaryote, transfection method of DNA ascalcium phosphate-precipitate, conventional mechanical procedures suchas microinjection, insertion of a plasmid encapsulated in red blood cellhosts or in liposomes, treatment of cells with agents such aslysophosphatidyl-choline or use of virus vectors, or the like may beused.

The level of production of a protein or polypeptide is governed by twomajor factors: the number of copies of its gene or DNA sequence encodingfor it within the cell and the efficiency with which these gene andsequence copies are transcribed and translated. Efficiencies oftranscription and translation (which together comprise expression) arein turn dependent upon nucleotide sequences, normally situated ahead ofthe desired coding sequence. These nucleotide sequences or expressioncontrol segpences define, inter alia, the location at which RNApolymerase interacts to initiate transcription (the promoter sequence)and at which ribosomes bind and interact with the mRNA (the product oftranscription) to initiate translation. Not all such expression controlsequences function with equal efficiency. It is thus of advantage toseparate the specific coding sequences for the desired protein fromtheir adjacent nucleotide sequences and fuse them instead to knownexpression control sequences so as to favor higher levels of expression.This having been achieved, the newly engineered DNA fragment may beinserted into a multicopy plasmid or a bacteriophage derivative in orderto increase the number of gene or sequence copies within the cell andthereby further improve the yield of expressed protein.

Several expression control sequences may be employed. These include theoperator, promoter and ribosome binding and interaction sequences(including sequences such as the Shine-Dalgarno sequences) of thelactose operon of E. coli ("the lac system"), the correspondingsequences of the tryptophan synthetase system of E. coli ("the trpsystem"), a fusion of the trp and lac promoter ("the tac system"), themajor operator and promoter regions of phage λ (O_(L) P_(L) and O_(R)P_(R')), and the control region of the phage fd coat protein. DNAfragments containing these sequences are excised by cleavage withrestriction enzymes from the DNA isolated from transducing phages thatcarry the lac or trp operons, or from the DNA of phage λ or fd. Thesefragments are then manipulated in order to obtain a limited populationof molecules such that the essential controlling sequences can be joinedvery close to, or in juxtaposition with, the initiation codon of thecoding sequence.

The fusion product is then inserted into a cloning vehicle fortransformation of the appropriate hosts and the level of antigenproduction is measured. Cells giving the most efficient expression maybe thus selected. Alternatively, cloning vehicles carrying the lac, trpor λ P_(L) control system attached to an initiation codon may beemployed and fused to a fragment containing a sequence coding for a gGprotein or polypeptide such that the gene or sequence is correctlytranslated from the initiation codon of the cloning vehicle.

The following examples and those infra are presented to help in thebetter understanding of the subject invention and for purposes ofillustration only. They are not to be construed as limiting theinvention in any manner.

EXAMPLE 1 Construction of Plasmid 19gGSE

Preparation of vector DNA. The pUC19 vector (described above) containsthe lac promoter followed by the alpha-peptide coding region ofbeta-galactosidase. Gene fragments that are subcloned into this vectormay be expressed as fusion proteins to the alpha-peptide.

The pUC19 vector plasmid was digested with EcoRI restriction enzyme andthen treated with calf intestine alkaline phosphatase. Anoligonucleotide adapter was synthesized to change the EcoRI overhang toan EagI overhang: ##STR2##

The lower oligonucleotide was kinased and then allowed to anneal withthe upper oligonucleotide. The adapter was then ligated with the EcoRIdigested pUC19 plasmid. After ligation, the plasmid containing theoligonucleotide adapter molecule was digested with SmaI restrictionenzyme. The plasmid DNA was separated from excess oligonucleotideadapter by gel isolation on a 1% Seaplaque® agarose gel. The agaroseslice containing the plasmid DNA was melted at 70° C., extracted twotimes with phenol, and precipitated with two volumes of 100% ethanol.

Preparation of the gG insert DNA. A plasmid containing the entire HSV-2gG gene was obtained from D. J. McGeoch (plasmid described previously).This plasmid was digested with restriction enzymes SspI and EagI. The1337 base pair fragment representing 92% of the gG unique sequence wasgel isolated on a 1% Seaplaque agarose gel as described above. Thisfragment was then ligated with the pUC19 plasmid containing theoligonucleotide adapter. (The SspI and SmaI sites are compatible forligation because both of these enzymes produce blunt ends.) The ligationproduced plasmid 19gGSE, which contains a portion of the gG uniquesequence fused in frame to the alpha-peptide coding region ofbeta-galactosidase. Expression is under the control of the lac promoter.E. coli strain JM103 was transformed with p19gGSE.

Preparation of E. coli lysates. Plasmid 19gGSE was additionallytransformed into E. coli strain CAG456. P19gGSE was tested forexpression of the gG/alpha-peptide fusion protein in both strains JM103and CAG456. For JM103, cells were grown until A550=0.750. The promoterwas then derepressed by the addition ofisopropyl-beta-D-thioglacto-pyranoside (IPTG) to give a finalconcentration of 1 mM. Samples were taken at certain time points duringthe cultures growth and prepared for Western blot analysis by pelletingcells, resuspending in Laemmli buffer (62.5 mM Tris, pH 6.81, 10%glycerol, 5% beta-mercaptoethanol, 2.3% SDS) at 0.03 A550 per microliterand boiled for 10 minutes. Ten microliter samples were run on proteingels and Western blotted as described.

In CAG456, the lac promoter behaves constitutively, so that derepressionwith IPTG is not necessary. Cells were pelleted, resuspended in Laemmlibuffer as described above for JM103, and analysed by Western blot.Lysates from both JM103 and CAG456 were tested for the presence of therecombinant gG protein in Western blot (see section below thereon fordetails) analysis using both HSV-2 culture confirmed patient sera and arabbit antibody made to HSV-2 (Dako). Faint bands corresponding to60,000 and 30,000 daltons were seen in CAG456 lysates. The theoreticalmolecular weight of the gG/alpha-peptide fusion protein is about 60,000daltons. The reactivity seen at 30,000 daltons could possibly be abreakdown product.

As the level of expression appeared significantly better in CAG456,probably because of lower levels of protease therein, the fusion proteinproduced in that strain was selected for further purification describedbelow in Example 3.

EXAMPLE 2 Construction of Plasmid TrpE/gG

The pPL-Lambda inducible expression vector is a coding vector which canbe purchased from Pharmacia (Piscataway, N.J.; Code No. 27-4946-01).(FIG. 5 is a diagram of that vector.) The pPL-Lambda vector contains 2BamHI sites. It is desirable to eliminate one of the BamHI sites bydeleting out a small SmaI fragment; to do so, the vector is digestedwith SmaI and religated, generating plasmid PLΔSma, which contains onlyone BamHI site.

Addition of TrpE to PLΔSma. The next step is the addition of the trpEleader sequence. The PLΔSma plasmid is first digested with SphI and thentreated with T₄ polymerase to provide a blunt end. The plasmid is thendigested with BamHI and treated with phosphatase.

The following oligo-adapter #1 containing the trpE leader issynthesized: ##STR3##

The upper strand of oligo-adapter #1 is kinased, allowed to anneal withthe lower strand, and then ligated with the BamHI/blunt PLΔSma plasmid,generating a vector with two blunt ends that contains the trpE leader.The plasmid is gel isolated.

Preparation of the gG Fragment. The McGeoch plasmid containing gG (asdescribed above in Example 1) is digested with SspI and EagI and treatedwith phosphatase. A 1337 base pair gG band is isolated from the gel andcan be represented as follows: ##STR4##

The following oligo-adapter (#2) is synthesized: ##STR5##

The upper strand thereof is kinased and annealed to the lower strand.Oligo-adapter #2 is then ligated with the gG fragment, generating a gGSspI/EcoRI fragment as follows: ##STR6##

Preparation of the gG/α-Peptide Fusion Fragment. The plasmid pUC19(described above in Example 1) is digested with BspHI and treated withDNA polymerase I-Klenow enzyme to effect blunt ends. The blunt-endedplasmid is treated with phosphatase and then digested with EcoRI.Isolated from the gel is a 448 base pair α-peptide fragment which isrepresented as follows: ##STR7##

The α-peptide fragment is ligated with the gG SspI/EcoRI fragment,generating the following gG/α-peptide blunt fragment: ##STR8##

That fragment is isolated from the gel, kinased and ligated with theblunt-ended trpE vector, generating plasmid trpE/gG. The plasmid ispreferably transformed into E. coli strain N4830 to producerecombinantly a fused protein containing a serologically active HSV-2 gGsegment. The E. coli N4830 strain has a lambda repressor which is heatsensitive; at higher temperatures, the repressor is deactivated, therebyderepressing the expression of the trpE/gG fused protein. In other E.coli strains not having such a lambda repressor, the trpE/gG proteinwould be produced constituitively.

Protein Purification

Example 3 is illustrative of a partial purification of the fused proteinproduced in accordance with Example 1.

EXAMPLE 3 Partial Purification

The E. coli pellet produced in accordance with Example 1 from E. colistrain CAG456 was resuspended in 50 mM Tris, pH 8.1, and lysozyme wasadded to 0.2 mg/mL. The suspension was kept on ice for one hour, thenfrozen at -70° C. for 2 hrs. After thawing, the lysate was brought to 2mM MgCl₂ and DNase I was added to 5 μg/mL. The lysate was kept on icefor 30 minutes, then spun at 10K rpm for 30 minutes at 4° C. The pellet(Pellet 1) was resuspended by sonication in 50 mM Tris, pH 8.1, 5 mMEDTA, 5 mM EGTA containing 1% NP-40 and 1% CHAPS. The resuspended pelletwas kept at room temperature for one hour, then centrifuged at 10K rpmfor 30 minutes at 22° C. Pellet 2 was resuspended in 7M guanidine-HCland dialyzed into 8M urea.

Further Purification

In order to purify the protein expressed in the host cell further,standard techniques of protein chemistry may be used. A process may bedeveloped by first experimenting with separating the expressed fusionprotein from contaminating host proteins by a series of extractions withdifferent detergents on sample aliquots. The detergents may be fromseveral different categories including anionic, cationic, nonionic andzwitterionic detergents. If a greater than 50/50 partition of theexpressed protein and contaminating host proteins is achieved, thedetergent providing the best partition may be selected as a preferredextractant. A preferred nonionic detergent for extracting the HSV-2 gGfused protein of this invention is NP-40 which was found to solubilizecontaminating proteins in Pellet 2 of Example 3 above.

An evaluation of the degree of purity of the expressed protein achievedin the detergent extraction is then made. If further purification isconsidered desirable, a series of chaotropic extractions at differentconcentrations may then be tried. Preferred chaotropes include 0 to 8Murea, 0 to 7M guanidine HCl, and 0 to 4M guanidine thiocyanate (SCN).Evaluation of the effects of such chaotropic extractions on the purityof the expressed protein is then made by SDS Page (sodium dodecylsulfate polyacrylamide gel electrophorests) and by Western blotting. Thepreferred chaotropes for purifying the recombinant HSV-2 gG proteins ofthis invention, based on their solubilizing effects on the contaminatingproteins of Pellet 2 of Example 3, are 4M guanidine SCN, 7M guanidineHCl and 8M urea, wherein 8M urea is more preferred.

It is preferred that once the expressed HSV-2 gG protein has beensolubilized using a chaotrope which is corrosive, viscous and/orexpensive, such as guanidine SCN or guanidine HCl, that such chaotopesbe removed before the next purification step, preferably by dialysis orby size exclusion chromatography.

The expressed HSV-2 gG protein may be further purified by conventionalmethods such as ion exchange chromatography. As many E. colicontaminating proteins are not charged or weakly charged at neutral pH,it is advantageous for the expressed protein of interest to have acharge at neutral pH in that such a charge differential between theexpressed protein and the E. coli proteins allows for good separationupon an ion exchange column. If the expressed protein has a negativecharge at neutral pH, anionic exchange chromatography is preferred;whereas if the expressed protein has a positive charge at neutral pH,cationic exchange chromatography is preferred. The expressed HSV-2 gGprotein produced according to Example 1 has been determined to have anet charge of +5 at neutral pH. Therefore, cationic exchangechromatography is considered a preferred purification step. It would befurther preferred that such cationic exchange chromatography beperformed at a slightly acidic pH. Although it would be preferable notto have to use chaotropes during chromatography, if a chaotrope isnecessary, urea would be a preferred chaotrope; for example theexpressed fusion protein purified partially in Example 3 can remain inthe 8M urea chaotrope during such chromatography.

Additional purification methods which may be useful are sizefractionation using molecular sieve chromatography, affinitychromatography, using for example, antibodies directed to the expressedHSV-2 gG protein, adsorption chromatography using non-specific supportsand also gel-supported electrophoresis, preferably SDS gelelectrophoresis.

In developing a protein purification process, it is desirable tominimize the number of purification steps and to maximize the recoveryand purification. A preferred process for purifying the expressed HSV-2gG proteins of this invention would be initially to solubilize thecontaminating proteins in the pellet with a nonionic detergent,preferably NP-40; then further extract the resulting pellet from thedetergent-solubilized materials with a chaotrope, preferably 8M urea;and then use ion exchange chromatography, preferably cationic exchangechromatography.

Example 4 represents a specific procedure performed to purify arepresentative expressed gG protein of this invention.

EXAMPLE 4

7.5 ml of Pellet 2 in 8M urea from Example 3 above was applied to aTSK-SP-5-PW cationic exchange column (7.5×75 mm; Biorad, Hercules,Calif.). Buffer A was 25 mM NaPO₄ at pH 5.0; and Buffer B was 1M NaCland 25 mM NAPO₄ at pH 5.0. From zero to 20 minutes, 100% of Buffer A wasapplied; and from 20 to 70 minutes, a 0-50% gradient of Buffer B wasapplied. The gG expressed protein was eluted at approximately aconcentration of 35% of Buffer B.

Epitopes

When preparing proteins or polypeptides for use as immunologicalreagents or as vaccines, it is usually desirable that the nucleotidesequence code for a protein or polypeptide that corresponds to one ormore epitopes of the natural HSV-2 gG protein. Also within the scope ofthis invention are synthetic and naturally produced polypeptides andproteins which contain epitopes of the HSV-2 gG protein as well as thecorresponding nucleotide sequences which encode such serologically andantigenically useful polypeptide and proteins. In this regard (referringto FIG. 2), suitable polypeptides and proteins are preferably selectedfrom the unique HSV-2 gG amino acid sequences, which include thefollowing amino acid sequences of the HSV-2 gG unique sequence which areconsidered to contain one or more epitopes: from about amino acid number45 to about amino acid number 70, from about amino acid number 95 toabout amino acid number 120, from about amino acid number 165 to aboutamino acid 230, from about amino acid number 305 to about amino acid330, and from about amino acid number 425 to about amino acid number450; wherein the preferred regions are from about 45 to about 70, fromabout 95 to about 120, from about 305 to about 330 and from about 425 toabout 450; and still more preferred are the regions from about 95 toabout 120 and from about 305 to about 330. Thus, the following aminoacid sequences are preferred according to this invention as are proteinsand polypeptides which comprise one or more of said amino acidsequences:

    H E P L G R S F L T G G L V L L A P P V R G F G A P        [SEQ. ID. NO.: 8];

    Q Y G G C R G G E P P S P K T C G S Y T Y T Y Q G G        [SEQ. ID. NO.: 9]; ##STR9##

    R T G R R L M A L T E D T S S D S P T S A P E K T P        [SEQ. ID. NO.: 11];

and

    P T S T H A T P R P T T P G P Q T T P P G P A T P G        [SEQ. ID. NO.: 12].

More preferred are the following amino acid sequences

    H E P L G R S F L T G G L V L L A P P V R G F G A P        [SEQ. ID. NO.: 8];

    Q Y G G C R G G E P P S P K T C G S Y T Y T Y Q G G        [SEQ. ID. NO.: 9];

    R T G R R L M A L T E D T S S D S P T S A P E K T P        [SEQ. ID. NO.: 11];

and

    P T S T H A T P R P T T P G P Q T T P P G P A T P G        [SEQ. ID. NO.: 12].

Still more preferred are the amino acid sequences:

    Q Y G G C R G G E P P S P K T C G S Y T Y T Y Q G G        [SEQ. ID. NO.: 9];

and

    R T G R R L M A L T E D T S S D S P T S A P E K T P        [SEQ. ID. NO.: 11];

The corresponding nucleotide sequences that code for such regions are asfollows, wherein the numbers used to identify such nucleotide sequencescorrespond to those in FIG. 1: from about nucleotide number 133 to about210; from about 283 to about 360; from about 493 to about 690; fromabout 913 to about 990, and from about 1273 to about 1350; wherein thepreferred sequences are those from about 133 to about 210, from about283 to about 360, from about 913 to about 990 and from about 1273 toabout 1350; and wherein the more preferred sequences are from about 283to about 360 and from about 913 to about 990.

Synthetic and Biologic Production of HSV-2 gG Protein and PolypeptideFragments Thereof

HSV-2 gG proteins and polypeptides of this invention may be formed notonly by recombinant means but also by synthetic and by other biologicmeans. Exemplary of other biologic means to prepare the desiredpolypeptide or protein is to subject to selective proteolysis a longergG polypeptide or protein containing the desired amino acid sequence;for example, the longer polypeptide or protein can be split withchemical reagents or with enzymes. Synthetic formation of thepolypeptide or protein requires chemically synthesizing the desiredchain of amino acids by methods well known in the art.

The portion of a longer polypeptide or protein which contains thedesired amino acids sequence can be excised by any of the followingprocedures:

(a) Digestion of the protein or longer polypeptide by proteolyticenzymes, specially those enzymes whose substrate specifically results incleavage of the protein or polypeptide at sites immediately adjacent tothe desired sequence of amino acids.

(b) Cleavage of the protein or polypeptide by chemical means. Particularbonds between amino acids can be cleaved by reaction with specificreagents. Examples include: bonds involving methionine are cleaved bycyanogen bromide: asparaginyl glycine bonds are cleaved byhydroxylamine; disulfide bonds between two cysteine residues are cleavedby reduction, e.g., with dithiothreitol.

(c) A combination of proteolytic and chemical changes. Of course, asindicated above, it should also be possible to clone a small portion ofthe DNA that codes for the synthetic peptide, resulting in theproduction of the peptide by the unicellular host.

The biologically or synthetically produced proteins and polypeptidesonce produced, may be purified by gel filtration, ion exchange or highpresure liquid chromatography, or other suitable means.

Chemical synthesis of polypeptides is described in the followingpublications: Merrifield et al., J. Am. Chem. Soc., 85:2149-2156 (1963);Kent et al., Synthetic Peptides in Biology and Medicine, 29 ff., edsAlitalo et al. (Elsevier Science Publishers 1985); Haug, ABL, 40-47(Jan/Feb. 1987); Andrews, Nature, 319:429-430 (Jan. 30, 1986); Kent,Biomedical Polymers, 213-242, eds. Goldberg et al. (Academic Press1980); Mitchell et al., J. Org. Chem., 43: 2845:2852 (1978); Tam et al.,Tet. Letters, 4033-4036 (1979); Mosjov et al., J. Org. Chem., 45:555-560 (1980); Tam et al. Tet Letters, 2851-2854 (1981); and Kent etal., Proceedings of the IV International Symposium on Methods of ProteinSequence Analysis (Brookhaven Press 1981).

The "Merrifield solid phase procedure" as described in theabove-mentioned publications can be used to build up the appropriatesequence of L-amino acids from the carboxyl terminal amino acid to theamino terminal amino acids. Starting with the appropriate carboxylterminal amino acid attached to an appropriate resin via chemicallinkage to a chloromethyl group, benzhydrylamine group, or otherreactive group of the resin, amino acids are added one by one using thefollowing procedure for each:

(a) Peptidyl resin is washed with methylene chloride;

(b) the resin is neutralized by mixing for 10 minutes at roomtemperature with 5% (v/v) diisoproplethylamine (or other hindered base)in methylene chloride;

(c) the resin is washed with methylene chloride:

(d) an amount of amino acid equal to six times the molar amount of thegrowing peptide chain is activated by combining it with one-half as manymoles of a carbodiimide, e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, for 10 minutes at 0° C., to form the symmetricanhydride of the amino acid. The amino acid used should be providedoriginally as the N-α-butyloxycarbonyl derivative, with side chainsprotected with benzyl esters (aspartic and glutamic acids), benzylethers (serine, threonine, cysteine, tyrosine), benzyl oxycarbonylgroups (lysine) or other protecting groups commonly used in peptidesynthesis:

(e) the activated amino acid is reacted with the peptidyl resin for 2hours at room temperature resulting in addition of the new amino acid tothe end of the growing peptide chain:

(f) the resin is washed with methylene chloride:

(g) The N-α-(butyloxycarbonyl) group is removed from the most recentlyadded amino acid by reacting with 30% (v/v) trifluoracetic acid inmethylene chloride for 30 minutes at room temperature.

(h) the resin is washed with methylene chloride;

(i) steps a through h are repeated until the required peptide sequencehas been constructed. The peptide is then removed from the resin andsimultaneously the side-chain protecting groups are removed, by reactingwith anhydrous hydrofluoric acid containing 10% (v/v) of anisole.Subsequently, the peptide can be purified by gel filtration, ionexchange, or high pressure liquid chromatography, or other suitablemeans.

Chemical synthesis can be carried out without a solid phase resin, inwhich case the synthetic reactions are performed entirely in solution.The reactions, and the final product, are otherwise essentiallyidentical.

Techniques of chemical peptide synthesis include using automatic peptidesynthesizers, employing commercially available protected amino acids;such synthesizers include, for example, Biosearch (San Rafael, Calif.)Models 9500 and 9600, Applied Biosystems Inc. (Foster City, Calif.)Model 430, and MilliGen (a division of Millipore Corp.) Model 9050.Further, one can manually synthesize up to about 25 polypeptides at atime by using Dupont's Ramp (Rapid Automated Multiple Peptide Syntheis).

The synthetic polypeptides according to this invention preferablycomprise one or more epitopes of the HSV-2 unique sequence gG,preferably as indicated above. It is possible to synthesize suchpolypeptides by attaching the amino acid sequence which defines anepitope (which can be from about three to about eight amino acids, moreusually from .about five to about eight amino acids) to at least threeamino acids flanking either side thereof. The three amino acids oneither side can be the same amino acids as in the natural unique gGsequence or could be other amino acids.

Testing for Serological Activity

Immunoblot Procedure. Serological reactivity of the recombinant gGprotein, prepared according to Example 1 and partially purifiedaccording to Example 3 was evaluated by Western immunoblot assay.

General background on the Western immunoblot assay technique can befound in: Towbin, et al., PNAS (USA), 76:4350 (1979); Towbin et al.,U.S. Pat. No. 4,452,901 (Jun. 5, 1984); Towbin, et al., J. Immunol.Methods, 72(2):313 (1984); and Bittner et al., Anal. Biochem., 102:459,(1980).

The specific procedure outlined below in Example 5 was used to test therecombinant unique sequence gG protein produced according to thisinvention. The protein was reactive with rabbit HSV-2 antiserum but notwith rabbit HSV-1 antiserum. [The rabbit antisera were purchased fromDako.] In addition, the recombinant gG protein reacted with a HSV-2 gGmonoclonal antibody obtained from N. Balachandran (University ofFlorida, Gainesville, Fla.). That anti-HSV-2 monoclonal antibody isdescribed in Balachandran et al., J. Virol., 44: 344-355 (1982).

Patient sera tested by immunoblot according to Example 5 (the resultsfor which are recorded in Table 2) were obtained from L. M. Frenkel(UCLA School of Medicine; Los Angeles, Calif.), C. Probet (StanfordUniversity; Palo Alto, Calif.), from J. Kettering (Loma Linda MedicalCenter, Loma Linda, Calif.) and Biomedical Resources (Pa.).

Thirty-seven out of 39 patient sera known to have antibody to HSV-2established by virus isolation, clinical history or by positivereactivity with native, glycosylated, full-length gG as assayed bySullender et al., supra were reactive in the immunoblot assay with thepartially purified recombinant, nonglycosylated, unique sequence gGprotein of this invention. None of the 19 patient sera that had onlyprior HSV-1 infection as established by clinical history or absence ofantibody reactivity with native HSV-2 gG as assayed by Sullender et al.,id showed reactivity in the immunoblot assay. None of the eight patientsera which were established to be free of either HSV-1 or HSV-2antibodies showed reactivity in the immunoblot assay.

Paired acute and convalescent sera from four patients similarly testedby immunoblot according to Example 5 (the results for which are recordedin Table 3) were obtained from the Department of Health Services--HealthProtection Division of Berkeley, Calif. Table 3 indicates that therepresentative unique gG sequence protein of this invention, partiallypurified according to Example 3, is useful in an immunoblot assay todiagnose whether a patient has had active HSV-2 infection. The darknessof the band on the Western blot indicating reactivity of a patient'santibodies to the unique sequence gG protein is directly proportional tothe patient's antibody titer. An increase in antibody titer from that ina patient's acute serum sample to that in the same patient'sconvalescent serum sample (taken 10 days later) is indicative of anactive infection of HSV-2. Table 3 indicates that all of the pairedserum samples of the three patients, who had been confirmed as havingHSV-2 by viral isolation and typing, were positive in the representativeserological assay of this invention; further, the titer rise shown fromthe acute to the convalescent samples indicated that all three patientshad active infections. Both of the paired sera samples of the onepatient, who had been confirmed to have a HSV-1 infection, registerednegative in the assay.

An advantage of the recombinant unique sequence gG of this inventionover the native gG of the Sullender et al. assay is its lack of epitopesto HSV-1. That advantage was demonstrated by the lack of reactivity therecombinant gG had with anti-HSV-1 rabbit polyclonal antibody andantibody from HSV-1 infected only patient sera. Thus, thecross-reactivity problem of the Sullender et al. assay using native,glycosylated, full-length gG in differentiating between HSV-1 and HSV-2antibodies, especially wherein antibodies to both serotypes are presentin patient sera, is obviated by the use of the recombinant uniquesequence gG proteins and polypeptides of this invention. An additionaladvantage of the recombinant gG is that it is much cheaper to producethan the native gG.

EXAMPLE 5 Immunoblot procedure

Pellet 2 of Example 3 was electrophoresed on 8% polyacrylamide slab gelsin the presence of SDS using the procedure of Laemmli. Proteins wereelectrophoretically transferred onto nitrocellulose for 60 minutes at200 mA using transfer buffer composed of 25 mM Tris-HCl, 192 mM glycine,and 20% methanol. The nitrocellulose was blocked for 20 minutes in 1Mglycine, 5% (w/v) nonfat dry milk, and 1% (w/v) ovalbumin, and was thenincubated with sera diluted 1/75 in blocking buffer at 4 degrees C.overnight. After three 3-minute rinses in PBST, the nitrocellulose wasincubated with HRP-labeled goat anti-human IgG in 10% FBS in PBST for 2hours at room temperature. The nitrocellulose was again rinsed anddeveloped in substrate-chromogene solution containing 0.2 mg/mL3,3'-diaminobenzidine -4HCl, 0.02% (w/v) NiCl₂, and 0.05% (w/v) H₂ O₂ in10 mM Tris-HCl, pH 7.5. The reaction was stopped by rinsing thenitrocellulose in water. The results of such assays are summarized inTables 2 and 3 below.

                  TABLE 2                                                         ______________________________________                                        Table 2. Serological reactivity of recombinant HSV-2-specific gG              with patient sera.                                                                             No. of                                                       Patient Group    Patients                                                                              % Positive.sup.a                                     ______________________________________                                        Prior HSV-2      39      95%                                                  infection.sup.b                                                               Prior HSV-1      19      0%                                                   Only.sup.c                                                                    No Prior HSV-1    8      0%                                                   or HSV-2.sup.d                                                                ______________________________________                                         .sup.a Positive reactivity determined by immunoblot assay using partially     purified recombinant HSV2-specific gG.                                        .sup.b Prior HSV2 infections established by virus isolation, clinical         history or by positive reactivity with native HSV2 gG as assayed by           Sullender et al., supra.                                                      .sup.c Prior HSV1 infection only established by clinical history or           absence of antibody reactivity with native HSV2 gG as assayed by Sullende     et al., supra.                                                                .sup.d No prior HSV1 or HSV2 infection established by the absence of HSV1     or HSV2 antibodies. (Plummer et al., supra.)                             

                  TABLE 3                                                         ______________________________________                                        Detection of recombinant HSV-2-specific gG antibody in                        acute/convalescent serum pairs.                                                        No. of Patients                                                      Type of HSV-2                                                                          with Paired                                                                              Acute Sera                                                                              Convalescent                                                                           Titer                                  Infection.sub.a                                                                        Samples    Samples.sub.b                                                                           Samples.sub.b                                                                          Rise.sub.c                             ______________________________________                                        HSV-2    3          all 3 either                                                                            all 3    Yes                                                        positive  strongly                                                            or weakly positive                                                            positive                                                  HSV-1    1          negative  negative No                                     ______________________________________                                         .sub.a The type of HSV infection was determined by the isolation and          typing the virus from the patients' lesions.                                  .sub.b An acute serum specimens was drawn from the patients at the time o     their first visit to the clinician. A second serum sample (the                convalescent sample) was drawn ten days later.                                .sub.c Demonstration of a rise in antibody levels to recombinant              HSV2-specific gG was done by immunoblot assay.                           

Diagnostic Tests for HSV-2

It is clear that the unique sequence HSV-2 gG proteins and polypeptidesof the instant invention may be used as diagnostic reagents for thedetection of HSV-2 type-specific antibodies. Polypeptides or proteinsdisplaying unique sequence HSV-2 gG antigenicity and the DNA sequenceswhich code therefor may be used in methods and kits designed to detectthe presence of type-specific antibodies in humans and thereforerecognize humans which have been infected by this virus.

For example, the unique sequence gG proteins and polypeptides producedby hosts transformed by recombinant DNA molecules of this invention canbe used in the formats of the immunological diagnostic tests currentlyavailable, that is, radioimmunoassay or ELISA (enzyme linkedimmunosorbent assay).

Preferably in one type of ELISA test, a microtiter plate is coated withunique sequence gG protein/polypeptide and to this is added a sample ofpatient's serum. After a period of incubation permitting any antibody tobind to the antigen, the plate is washed and a preparation of anti-humanantibodies, raised in a laboratory animal, and which are linked to anenzyme is added, incubated to allow reaction to take place, and theplate is then rewashed. Thereafter, enzyme substrate is added to themicrotiter plate and incubated for a period of time to allow the enzymeto work on the substrate, and the adsorbance of the final preparation ismeasured. A large change in absorbance indicates a positive result.

It is also apparent to one of ordinary skill that a diagnostic assay forHSV-2 using polyclonal or monoclonal antibodies to the HSV-2 uniquesequence gG proteins and polypeptides of the instant invention may beused to detect the presence of HSV-2. In one embodiment a competitionimmunoassay is used wherein the antigenic substance, in this case HSV-2,in a vesicle sample competes with a known quantity of labelled antigen,in this case labelled unique sequence HSV-2 gG proteins andpolypeptides, for a limited quantity of antibody binding sites. Thus,the amount of labelled antigen bound to the antibody is inverselyproportional to the amount of antigen in the sample. In anotherembodiment, an immunometric assay may be used wherein a labelledantibody to a HSV-2 unique sequence protein or polypeptide is used. Insuch an assay, the amount of labelled antibody which complexes with theantigen-bound antibody is directly proportional to the amount of antigen(HSV-2) in the vesicle sample. In a simple yes/no assay to determinewhether HSV-2 is present in vesicle specimens, the solid support istested to detect the presence of labelled antibody. In anotherembodiment, monoclonal antibodies to the HSV-2 unique sequence gGproteins or polypeptides may be used in an immunometric assay. Suchmonoclonal antibodies may be obtained by methods well known in the art,particularly the process of Kohler and Milstein reported in Nature,256:495-497 (1975). Example 6, immediately below is representative ofsuch an immunometric assay.

EXAMPLE 6 Immunometric Assay for HSV-2

Rabbit polyclonal antibody produced against HSV-2 unique sequence gGprotein and/or polypeptides is prepared. Duplicate samples are run inwhich 100 ul of a suspension of such antibody immobilized on agaroseparticles is mixed with 100 ul of serum and 100 ul of soluble ¹²⁵I-labelled antibody produced against HSV-2 unique sequence proteinand/or polypeptide. This mixture is allowed to incubate for specifiedtimes ranging from one quarter hour to twenty-four hours. Following theincubation period, the agarose particles are washed by addition ofbuffer and then centrifuged. After removal of the washing liquid byaspiration, the resulting pellet of agarose particles is then countedfor bound ¹²⁵ I-labelled antibody. The counts obtained for each of thecomplexes can then be compared to the control sample.

Such diagnostic methods can be embodied in test kits to assay for HSV-2type-specific antibodies in human bodily fluids wherein such test kitscan comprise (a) a solid phase coated with recombinantly producednonglycosylated or glycosylated proteins encoded by the unique sequenceof HSV-2 gG or fragments thereof or with synthetically producedpolypeptides that have the same or substantially the same amino acidsequence as those encoded by the unique sequence of HSV-2 gG or portionsthereof or biologically produced gG unique sequence polypeptide and/orproteins; and (b) a detection means. Test kits designed to detect HSV-2itself can further comprise antibodies, preferably monoclonalantibodies, to the HSV-2 unique sequence proteins/polypeptides.

Suitable detection means include the use of labels such asradionuclides, enzymes, fluorescers, chemiluminescers, enzyme substratesor co-factors, enzyme inhibitors, particles, dyes and the like. Suchlabeled reagents may be used in a variety of well known assays, such asradioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescentimmunoassays, and the like. See, for example, U.S. Pat. Nos. 3,766,162;3,791,932; 3,817,837; and 4,233,402.

Antibodies to gG

Antibodies to the recombinant, synthetic or natural HSV-2 gG proteinsand polypeptides, preferably to the recombinant, synthetic or naturalunique sequence gG proteins/polypeptides of this invention, have use notonly for diagnostic assays but also for affinity purification of gGproteins/polypeptides and for therapeutic use by procedures of passiveimmunization. When the antibodies are used therapeutically for passiveimmunization or in diagnostic assays, it is preferred that they be tothe unique sequence gG proteins/polypeptides of this invention.

Vaccines

As indicated above and shown in Table 2, 95% of the tissue cultureconfirmed HSV-2 positive sera contained antibodies to the representativeunique specific recombinant gG protein of this invention. This datastrongly suggest that the HSV-2 unique sequence gG proteins andpolypeptides of this invention would be immunogenic in humans.

An advantage of using unique sequence gG proteins and/or polypeptides asvaccines against HSV-2 resides in their demonstrated lack ofcross-reactivity with antibodies to HSV-1. More people have been exposedto HSV-1 than HSV-2, and a substantial number of people have antibodiesto HSV-1. Adverse reactions may occur upon the introduction of a vaccinefor HSV-2, such as full-length glycosylated gG, which has epitopes thatare not unique to HSV-2 and which may react with the pre-existing HSV-1antibodies. Such adverse reactions, for example, vaccine reactions suchas immune complex diseases or anaphylactic shock, are not anticipated asa problem wherein unique sequence gG proteins and/or polypeptides areemployed as vaccines in that they are not cross-reactive with antibodiesto HSV-1.

It will be readily appreciated that the HSV-2 unique sequence gGproteins and polypeptides of this invention can be incorporated intovaccines capable of inducing protective immunity against HSV-2.Preferably, said HSV-2 unique sequence gG proteins and polypeptides arethose containing the amino acid sequences noted above as encompassingepitopes of the unique sequence. Polypeptides may be- synthesized orprepared recombinantly or otherwise biologically, to comprise one ormore amino acid sequences corresponding to one or more epitopes of theHSV-2 unique sequence gG either in monomeric or multimeric form. Thesepolypeptides may then be incorporated into vaccines capable of inducingprotective immunity against HSV-2. Techniques for enhancing theantigenicity of such polypeptides include incorporation into amultimeric structure, binding to a highly immunogenic protein carrier,for example, keyhole limpet hemocyanin (KLH), or diphtheria toxoid, andadministration in combination with adjuvants or any other enhancers ofimmune response. In addition, the vaccine composition may compriseantigens to provide immunity against other diseases in addition toHSV-2.

An amino acid sequence corresponding to an epitope of HSV-2 uniquesequence gG either in monomeric or multimeric form may be obtained bychemical synthetic means or by purification from biological sourcesincluding genetically modified microorganisms or their culture media.[See Lerner, "Synthetic Vaccines", Sci. Am. 248(2):66-74 (1983).] Thepolypeptide may be combined in an amino acid sequence with otherpolypeptides including fragments of other proteins, as for example, whensynthesized as a fusion protein, or linked to other antibenic ornon-antigenic polypeptides of synthetic or biological origin.

The term "corresponding to an epitope of a HSV-2 unique sequence gG"will be understood to include the practical possibility that, in someinstances, amino acid sequence variations of naturally occurring proteinand polypeptide may be antigenic and confer protective immunity againstHSV-2. Possible sequence variations include, without limitation, aminoacid substitutions, extensions, deletions, interpolations andcombinations thereof. Such variations fall within the contemplated scopeof the invention provided the protein or polypeptide containing them isantigenic and antibodies elicited by such polypeptide or proteincross-react with naturally occurring HSV-2 unique sequence gG proteinsand polypeptides to an extent sufficient to provide protective immunitywhen administered as a vaccine.

Such vaccine compositions will be combined with a physiologicallyacceptable medium, including immunologically acceptable diluents andcarriers as well as commonly employed adjuvants such as Freund'sComplete Adjuvant, saponin, alum, and the like. Administration would bein immunologically effective amounts of the HSV-2 unique sequence gGproteins or polypeptides, preferably in quantities providing unit doesof from 0.01 to 10.0 micrograms of immunologically active uniquesequence gG protein or polypeptide per kilogram of the recipient's bodyweight. Total protective doses may range from 0.1 to about 100micrograms of antigen.

Routes of administration, antigen dose, number and frequency ofinjections are all matters of optimization within the scope of ordinaryskill in the art particularly in view of the fact that there isexperience in the art in providing protective immunity by the injectionof other related antigens to provide immunity in other viral infections.[See Wise et al., "Herpes Simplex Virus Vaccines" J. Inf. Dis.,136:706-711 (1977).] It is anticipated that the principal value ofproviding immunity to HSV-2 infection will be for those individuals whohave had no previous exposure to HSV-2. It is also anticipated thattemporary immunity for infants may be provided by immunization ofmothers during pregnancy.

DNA Probes

The unique sequence of HSV-2 gG and fragments thereof are useful as DNAprobes which are specific for HSV-2. Said DNA probes are at least 14nucleotides long and usually from about 20 to about 70 nucleotides inlength. Specific examples of DNA probes from the unique sequence ofHSV-2 gG include the nucleic acid sequence from about nucleotide 45 toabout nucleotide 1386 of FIGS. 1A and 1B (cloned according to Example 1)and more preferably fragments thereof. Said DNA probes are purified andisolated from contaminating materials according to methods well known inthe art.

Conclusion

This invention provides for rapid diagnostic tests that are currentlyneeded by clinicians, especially by obstetricians, to diagnose bothasymptomatic and symptomatic HSV-2 infections. It may be seen, further,that the recombinantly, synthetically or biologically produced proteinsand polypeptides provided by this invention can serve not only asdiagnostic reagents but also as the basis for vaccines to protectagainst HSV-2. The invention still further provides for antibodies thatcan be used both therapeutically and diagnostically in regard to HSV-2infections. Still further, this invention provides for DNA probesspecific for HSV-2 DNA.

It is understood that the hybrid micro-organisms, recombinant DNAmolecules and proteins/polypeptides and methods applicable to them ofthis invention are not limited to those described in the preferredembodiments above. The hybrid organisms, recombinant DNA molecules andprotein/polypeptides may be modified during production or subsequentlyby known methods to good advantage. For example, more efficient controlsequences may be used for transcription of the HSV-2 gG sequences,mutations to reduce the synthesis of undesired products may beintroduced, the protease levels in the host cells may be reduced,thermo-inducible lysogens containing the HSV-2 gG sequences may beintegrated into the host chromosome or other modifications andprocedures may be carried out to increase the number of sequence copiesin the cell or to increase the cell's productivity in producing thedesired protein/polypeptide.

Various modifications of the invention in addition to those shown anddescribed herein will become apparent to those in the art from theforegoing description. Such modifications are intended to be within thescope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1461 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CAACAGCGATGTTGTTTTCCCGGGAGGTTCCCCCGTGGCTCAATATTGTTATGCCTATCC60                CCGGTTGGACGATCCCGGGCCCTTGGGTTCCGCGGACGCCGGGCGGCAAGACCTGCCCCG120               GCGCGTCGTCCGTCACGAGCCCCTGGGCCGCTCGTTCCTCACGGGGGGGCTGGTTTTGCT180               GGCGCCGCCGGTACGCGGATTTGGCGCACCCAACGCAACGTATGCGGCCCGTGTGACGTA240               CTACCGGCTCACCCGCGCCTGCCGTCAGCCCATCCTCCTTCGGCAGTATGGAGGGTGTCG300               CGGCGGCGAGCCGCCGTCCCCAAAGACGTGCGGGTCGTACACGTACACGTACCAGGGCGG360               CGGGCCTCCGACCCGGTACGCTCTCGTAAATGCTTCCCTGCTGGTGCCGATCTGGGACCG420               CGCCGCGGAGACATTCGAGTACCAGATCGAACTCGGCGGCGAGCTGCACGTGGGTCTGTT480               GTGGGTAGAGGTGGGCGGGGAGGGCCCCGGCCCCACCGCCCCCCCACAGGCGGCGCGTGC540               GGAGGGCGGCCCGTGCGTCCCCCCGGTCCCCGCGGGCCGCCCGTGGCGCTCGGTGCCCCC600               GGTATGGTATTCCGCCCCCAACCCCGGGTTTCGTGGCCTGCGTTTCCGGGAGCGCTGTCT660               GCCCCCACAGACGCCCGCCGCCCCCAGCGACCTACCACGCGTCGCTTTTGCTCCCCAGAG720               CCTGCTGGTGGGGATTACGGGCCGCACGTTTATTCGGATGGCACGACCCACGGAAGACGT780               CGGGGTCCTGCCGCCCCATTGGGCCCCCGGGGCCCTAGATGACGGTCCGTACGCCCCCTT840               CCCACCCCGCCCGCGGTTTCGACGCGCCCTGCGGACAGACCCCGAGGGGGTCGACCCCGA900               CGTTCGGGCCCCCCGAACCGGGCGGCGCCTCATGGCCTTGACCGAGGACACGTCCTCCGA960               TTCGCCTACGTCCGCTCCGGAGAAGACGCCCCTCCCTGTGTCGGCCACCGCCATGGCACC1020              CTCAGTCGACCCAAGCGCGGAACCGACCGCCCCCGCAACCACTACTCCCCCCGACGAGAT1080              GGCCACACAAGCCGCAACGGTCGCCGTTACGCCGGAGGAAACGGCAGTCGCCTCCCCGCC1140              CGCGACTGCATCCGTGGAGTCGTCGCCACTCCCCGCCGCGGCGGCGGCAACGCCCGGGGC1200              CGGGCACACGAACACCAGCAGCGCCTCCGCAGCGAAAACGCCCCCCACCACACCAGCCCC1260              CACGACCCCCCCGCCCACGTCTACCCACGCGACCCCCCGCCCCACGACTCCGGGGCCCCA1320              AACAACCCCTCCCGGACCCGCAACCCCGGGTCCGGTGGGCGCCTCCGCCGCGCCCACGGC1380              CGATTCCCCCCTCACCGCCTCGCCCCCCGCTACCGCGCCGGGGCCCTCGGCCGCCAACGT1440              TTCGGTCGCCGCGACCACCGC1461                                                     (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 486 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      AsnSerAspValValPheProGlyGlySerProValAlaGlnTyrCys                              151015                                                                        TyrAlaTyrProArgLeuAspAspProGlyProLeuGlySerAlaAsp                              202530                                                                        AlaGlyArgGlnAspLeuProArgArgValValArgHisGluProLeu                              354045                                                                        GlyArgSerPheLeuThrGlyGlyLeuValLeuLeuAlaProProVal                              505560                                                                        ArgGlyPheGlyAlaProAsnAlaThrTyrAlaAlaArgValThrTyr                              65707580                                                                      TyrArgLeuThrArgAlaCysArgGlnProIleLeuLeuArgGlnTyr                              859095                                                                        GlyGlyCysArgGlyGlyGluProProSerProLysThrCysGlySer                              100105110                                                                     TyrThrTyrThrTyrGlnGlyGlyGlyProProThrArgTyrAlaLeu                              115120125                                                                     ValAsnAlaSerLeuLeuValProIleTrpAspArgAlaAlaGluThr                              130135140                                                                     PheGluTyrGlnIleGluLeuGlyGlyGluLeuHisValGlyLeuLeu                              145150155160                                                                  TrpValGluValGlyGlyGluGlyProGlyProThrAlaProProGln                              165170175                                                                     AlaAlaArgAlaGluGlyGlyProCysValProProValProAlaGly                              180185190                                                                     ArgProTrpArgSerValProProValTrpTyrSerAlaProAsnPro                              195200205                                                                     GlyPheArgGlyLeuArgPheArgGluArgCysLeuProProGlnThr                              210215220                                                                     ProAlaAlaProSerAspLeuProArgValAlaPheAlaProGlnSer                              225230235240                                                                  LeuLeuValGlyIleThrGlyArgThrPheIleArgMetAlaArgPro                              245250255                                                                     ThrGluAspValGlyValLeuProProHisTrpAlaProGlyAlaLeu                              260265270                                                                     AspAspGlyProTyrAlaProPheProProArgProArgPheArgArg                              275280285                                                                     AlaLeuArgThrAspProGluGlyValAspProAspValArgAlaPro                              290295300                                                                     ArgThrGlyArgArgLeuMetAlaLeuThrGluAspThrSerSerAsp                              305310315320                                                                  SerProThrSerAlaProGluLysThrProLeuProValSerAlaThr                              325330335                                                                     AlaMetAlaProSerValAspProSerAlaGluProThrAlaProAla                              340345350                                                                     ThrThrThrProProAspGluMetAlaThrGlnAlaAlaThrValAla                              355360365                                                                     ValThrProGluGluThrAlaValAlaSerProProAlaThrAlaSer                              370375380                                                                     ValGluSerSerProLeuProAlaAlaAlaAlaAlaThrProGlyAla                              385390395400                                                                  GlyHisThrAsnThrSerSerAlaSerAlaAlaLysThrProProThr                              405410415                                                                     ThrProAlaProThrThrProProProThrSerThrHisAlaThrPro                              420425430                                                                     ArgProThrThrProGlyProGlnThrThrProProGlyProAlaThr                              435440445                                                                     ProGlyProValGlyAlaSerAlaAlaProThrAlaAspSerProLeu                              450455460                                                                     ThrAlaSerProProAlaThrAlaProGlyProSerAlaAlaAsnVal                              465470475480                                                                  SerValAlaAlaThrThr                                                            485                                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1342 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      ATTGTTATGCCTATCCCCGGTTGGACGATCCCGGGCCCTTGGGTTCCGCGGACGCCGGGC60                GGCAAGACCTGCCCCGGCGCGTCGTCCGTCACGAGCCCCTGGGCCGCTCGTTCCTCACGG120               GGGGGCTGGTTTTGCTGGCGCCGCCGGTACGCGGATTTGGCGCACCCAACGCAACGTATG180               CGGCCCGTGTGACGTACTACCGGCTCACCCGCGCCTGCCGTCAGCCCATCCTCCTTCGGC240               AGTATGGAGGGTGTCGCGGCGGCGAGCCGCCGTCCCCAAAGACGTGCGGGTCGTACACGT300               ACACGTACCAGGGCGGCGGGCCTCCGACCCGGTACGCTCTCGTAAATGCTTCCCTGCTGG360               TGCCGATCTGGGACCGCGCCGCGGAGACATTCGAGTACCAGATCGAACTCGGCGGCGAGC420               TGCACGTGGGTCTGTTGTGGGTAGAGGTGGGCGGGGAGGGCCCCGGCCCCACCGCCCCCC480               CACAGGCGGCGCGTGCGGAGGGCGGCCCGTGCGTCCCCCCGGTCCCCGCGGGCCGCCCGT540               GGCGCTCGGTGCCCCCGGTATGGTATTCCGCCCCCAACCCCGGGTTTCGTGGCCTGCGTT600               TCCGGGAGCGCTGTCTGCCCCCACAGACGCCCGCCGCCCCCAGCGACCTACCACGCGTCG660               CTTTTGCTCCCCAGAGCCTGCTGGTGGGGATTACGGGCCGCACGTTTATTCGGATGGCAC720               GACCCACGGAAGACGTCGGGGTCCTGCCGCCCCATTGGGCCCCCGGGGCCCTAGATGACG780               GTCCGTACGCCCCCTTCCCACCCCGCCCGCGGTTTCGACGCGCCCTGCGGACAGACCCCG840               AGGGGGTCGACCCCGACGTTCGGGCCCCCCGAACCGGGCGGCGCCTCATGGCCTTGACCG900               AGGACACGTCCTCCGATTCGCCTACGTCCGCTCCGGAGAAGACGCCCCTCCCTGTGTCGG960               CCACCGCCATGGCACCCTCAGTCGACCCAAGCGCGGAACCGACCGCCCCCGCAACCACTA1020              CTCCCCCCGACGAGATGGCCACACAAGCCGCAACGGTCGCCGTTACGCCGGAGGAAACGG1080              CAGTCGCCTCCCCGCCCGCGACTGCATCCGTGGAGTCGTCGCCACTCCCCGCCGCGGCGG1140              CGGCAACGCCCGGGGCCGGGCACACGAACACCAGCAGCGCCTCCGCAGCGAAAACGCCCC1200              CCACCACACCAGCCCCCACGACCCCCCCGCCCACGTCTACCCACGCGACCCCCCGCCCCA1260              CGACTCCGGGGCCCCAAACAACCCCTCCCGGACCCGCAACCCCGGGTCCGGTGGGCGCCT1320              CCGCCGCGCCCACGGCCGATTC1342                                                    (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 447 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CysTyrAlaTyrProArgLeuAspAspProGlyProLeuGlySerAla                              151015                                                                        AspAlaGlyArgGlnAspLeuProArgArgValValArgHisGluPro                              202530                                                                        LeuGlyArgSerPheLeuThrGlyGlyLeuValLeuLeuAlaProPro                              354045                                                                        ValArgGlyPheGlyAlaProAsnAlaThrTyrAlaAlaArgValThr                              505560                                                                        TyrTyrArgLeuThrArgAlaCysArgGlnProIleLeuLeuArgGln                              65707580                                                                      TyrGlyGlyCysArgGlyGlyGluProProSerProLysThrCysGly                              859095                                                                        SerTyrThrTyrThrTyrGlnGlyGlyGlyProProThrArgTyrAla                              100105110                                                                     LeuValAsnAlaSerLeuLeuValProIleTrpAspArgAlaAlaGlu                              115120125                                                                     ThrPheGluTyrGlnIleGluLeuGlyGlyGluLeuHisValGlyLeu                              130135140                                                                     LeuTrpValGluValGlyGlyGluGlyProGlyProThrAlaProPro                              145150155160                                                                  GlnAlaAlaArgAlaGluGlyGlyProCysValProProValProAla                              165170175                                                                     GlyArgProTrpArgSerValProProValTrpTyrSerAlaProAsn                              180185190                                                                     ProGlyPheArgGlyLeuArgPheArgGluArgCysLeuProProGln                              195200205                                                                     ThrProAlaAlaProSerAspLeuProArgValAlaPheAlaProGln                              210215220                                                                     SerLeuLeuValGlyIleThrGlyArgThrPheIleArgMetAlaArg                              225230235240                                                                  ProThrGluAspValGlyValLeuProProHisTrpAlaProGlyAla                              245250255                                                                     LeuAspAspGlyProTyrAlaProPheProProArgProArgPheArg                              260265270                                                                     ArgAlaLeuArgThrAspProGluGlyValAspProAspValArgAla                              275280285                                                                     ProArgThrGlyArgArgLeuMetAlaLeuThrGluAspThrSerSer                              290295300                                                                     AspSerProThrSerAlaProGluLysThrProLeuProValSerAla                              305310315320                                                                  ThrAlaMetAlaProSerValAspProSerAlaGluProThrAlaPro                              325330335                                                                     AlaThrThrThrProProAspGluMetAlaThrGlnAlaAlaThrVal                              340345350                                                                     AlaValThrProGluGluThrAlaValAlaSerProProAlaThrAla                              355360365                                                                     SerValGluSerSerProLeuProAlaAlaAlaAlaAlaThrProGly                              370375380                                                                     AlaGlyHisThrAsnThrSerSerAlaSerAlaAlaLysThrProPro                              385390395400                                                                  ThrThrProAlaProThrThrProProProThrSerThrHisAlaThr                              405410415                                                                     ProArgProThrThrProGlyProGlnThrThrProProGlyProAla                              420425430                                                                     ThrProGlyProValGlyAlaSerAlaAlaProThrAlaAspSer                                 435440445                                                                     (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GGCCGATTCG10                                                                  (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      CTAAGCTTAA10                                                                  (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      GATCCAGGAAATACTTACATATGAAAGCTATCTTCGTTCTGAAAGGTTCTCTGGACCGTG60                ACCCGGAATTCACCATGGATCCCC84                                                    (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      HisGluProLeuGlyArgSerPheLeuThrGlyGlyLeuValLeuLeu                              151015                                                                        AlaProProValArgGlyPheGlyAlaPro                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      GlnTyrGlyGlyCysArgGlyGlyGluProProSerProLysThrCys                              151015                                                                        GlySerTyrThrTyrThrTyrGlnGlyGly                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GlyGlyGluGlyProGlyProThrAlaProProGlnAlaAlaArgAla                              151015                                                                        GluGlyGlyProCysValProProValProAlaGlyArgProTrpArg                              202530                                                                        SerValProProValTrpTyrSerAlaProAsnProGlyPheArgGly                              354045                                                                        LeuArgPheArgGluArgCysLeuProProGlnThrProAlaAlaPro                              505560                                                                        SerAsp                                                                        65                                                                            (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     ArgThrGlyArgArgLeuMetAlaLeuThrGluAspThrSerSerAsp                              151015                                                                        SerProThrSerAlaProGluLysThrPro                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     ProThrSerThrHisAlaThrProArgProThrThrProGlyProGln                              151015                                                                        ThrThrProProGlyProAlaThrProGly                                                2025                                                                          __________________________________________________________________________

What we claim is:
 1. A recombinant DNA molecule comprising a HSV gG2nucleotide sequence encoding an amino acid sequence consisting of SEQ IDNO: 4, wherein the only gG2 nucleotide sequence present is that whichencodes the polypeptide of SEQ ID NO:
 4. 2. The recombinant DNA moleculeof claim 1 wherein the nucleotide sequence consists of SEQ ID NO:
 3. 3.A unicellular host which is either prokaryotic or eukaryotic which istransformed with the recombinant DNA molecule of claim
 1. 4. Theunicellular host according to claim 2 wherein the recombinant DNAmolecule is a recombinant cloning vehicle comprising a first and secondrestriction endonuclease recognition site, said DNA sequence beinginserted between said first and second restriction sites.
 5. Theunicellular host according to claim 3 wherein said host is E. coli,Pseudomonas, Bacillus yeast, fungi, an animal cell, an insect cell, or aplant cell.
 6. The unicellular host according to claim 5 which is an E.coli strain.
 7. A method for producing the unique sequence HSV-2glycoprotein G protein comprising the steps of:(a) transforming aunicellular host with a recombinant DNA molecule of claim 1, (b)culturing said transformed host so that said unique sequence HSV-2glycoprotein G protein is expressed; and (c) extracting and isolatingsaid unique sequence HSV-2 glycoprotein G protein.
 8. A recombinant DNAmolecule comprising a sequence which encodes a fragment of the uniquesequence HSV-2 glycoprotein G protein, wherein said sequence is selectedfrom the group consisting of nucleotide 133 to 210, 283 to 360, 493 to690, 913 to 990, and 1273 to 1350 of SEQ ID NO: 1, wherein said sequenceis operatively linked to an expression control sequence in said DNAmolecule.
 9. The recombinant DNA molecule according to claim 8 whereinsaid nucleotide sequence is 133 to 210, 283 to 360, 913 to 990 or 1273to 1350 of SEQ ID NO:
 1. 10. The recombinant DNA molecule according toclaim 9 wherein said nucleotide sequence is 283 to 360 or 913 to 990 ofSEQ ID NO:
 1. 11. A unicellular host which is either prokaryotic oreukaryotic which is transformed with the recombinant DNA molecule ofclaim
 8. 12. The unicellular host according to claim 11 wherein therecombinant DNA molecule is a recombinant cloning vehicle comprising afirst and second restriction endonuclease recognition site, said DNAsequence being inserted between said first and second restriction sites.13. The unicellular host according to claim 11 wherein said host is E.coli, Pseudomonas, Bacillus, yeast, fungi, an animal cell, an insectcell, or a plant cell.
 14. The unicellular host according to claim 13which is an E. coli strain.
 15. A method for producing a fragment of theunique sequence HSV-2 glycoprotein G protein comprising the steps of:(a)transforming a unicellular host with a recombinant DNA molecule of claim8; (b) culturing said transformed host so that said unique sequenceHSV-2 glycoprotein G protein is expressed; and (c) extracting andisolating said unique sequence HSV-2 glycoprotein G protein.
 16. Apurified and isolated DNA molecule which codes for the unique sequenceHSV-2 glycoprotein G protein which has the amino acid sequence fromamino acid 16 to 462 designated as SEQ. ID. No. 4, wherein said DNAconsists of the nucleotide sequence designated as SEQ. ID. No.
 3. 17. Apurified and isolated DNA molecule which codes for a fragment of theunique sequence HSV-2 glycoprotein G protein selected from the groupconsisting of nucleotide 133 to 210, 283 to 360, 493 to 690, 913 to 990,and 1273 to 1350 of SEQ ID NO: 1.